Cytochromes P-450 are key enzymes in the biosynthesis of steroids in the adrenal, testis, ovary, and placenta. We propose a comprehensive study of the structure-function relationships in these enzymes. One focus of our studies will be aromatase, the enzyme responsible for the conversion of androstenedione to estrone and testosterone to estradiol. We have purified and raised antibodies to human placental aromatase and have cloned a cDNA for this enzyme. The human cDNA probe has been used to pick up a clone for the rat enzyme. The rat probe will be used to study aromatase mRNA distribution in rat tissues for adult and neonatal rats. The adult rats will be treated with aromatase inhibitors to study their possible affect on mRNA levels. In neonates we will study mRNA levels in the brains of males and females before and after birth with and without inhibitors. These studies should provide new insight to the role of aromatase in neonatal imprinting on steroid liver metabolism. The metabolism of the aromatase inhibitor 4- hydroxyandrostenedione will also be studied by our collaborator, Wayne Levin at the Roche Institute of Molecular Biology. Bromoacetoxy derivatives of androstenedione will be used to probe the active site of purified aromatase. We will raise monoclonal antibodies to human placental aromatase which will be used to measure aromatase levels in rat tissues, and in a separate study, aromatase levels in breast cancer patients. We will purify and characterize bovine and rate 3-beta-hydroxysteroid dehydrogenase /steroid 5-ene-4-ene isomerase. This enzyme plays a key role in the conversion of pregnenolone to progesterone. Very little is known about the structure or dual enzymatic role of this key steroidogenic enzyme. We will raise antibodies to this enzyme to study its distribution in rat adrenal, testis, and ovaries, and perform active site studies on the NAD+-binding site with FSBA.